Hypocretin Modulation Of Synaptic Activity

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3.1. Hypocretin Increases Transmitter Release by Activating Receptors on Axon Terminals

Synaptic activity in both cultures and brain slices is enhanced by hypocretin (Figs. 7 and 8). This could be due to activation of receptors on the cell body, or to receptors on axonal boutons, or both. To address the question of whether hypocretin could act directly on axon terminals, action potential-mediated synaptic activity was blocked with TTX. Even when spikes are blocked, many axons continue to release neurotransmitters, although at a lower level, detected as miniature postsynaptic currents (mPSCs) or potentials. When hypocretin was added to cultures of hypothalamic neurons, increases in spontaneous excitatory (Fig. 7) and inhibitory (Fig. 8A) synaptic activity were found; an increase in the frequency of both gluta-mate-mediated m excitatory (E)PSCs and GABA-dependent m inhibitory (I)PSCs was also detected. In both cases, the frequency of miniature release events was increased, with no rise in the amplitude cumulative probability (Fig. 7B and C). This suggests that the increased synaptic frequency was not owing to an enhanced receptor response in the postsynaptic neuron, but rather was due to an increased release probability from both glutamate and GABA axon terminals (14). In parallel studies in the nucleus of the solitary tract, hypocretin increased the frequency of mEPSCs but not IPSCs (40); in this part of the brain, hypocretin appeared to selectively activate release from excitatory axons, but not from inhibitory axons. This suggests that hypocretin is not indiscriminantly excitatory, but rather can selectively activate subsets of cells.

In studies of responses in the prefrontal cerebral cortex, hypocretin increased release of glutamate from presumptive thalamocortical axons by a mechanism dependent on TTX-sensitive spikes (41). These data suggest that spikes may be initiated in the presynaptic boutons and that hypocretin may depolarize the boutons sufficiently to generate a spike leading to glutamate release and a postsynaptic response, even in the absence of the bouton's parent cell body.

Recently, a novel mechanism has been identified by which hypocretin may modulate glu-tamatergic transmission, based on the activation of retrograde endocannabinoid signaling. Haj-Dahmane and Shen (55) found that under some conditions, hypocretin reduced the release of glutamate from presynaptic axons onto serotonin neurons in the dorsal raphe. Unexpectedly, these hypocretin actions were abolished when postsynaptic serotonin cells were loaded with GDP^S (an inhibitor of G proteins) via the patch pipet, suggesting that hypocre-tin acted on its postsynaptic receptors and indirectly evoked a decrease in the release of glutamate from presynaptic terminals. The selective cannabinoid receptor 1 (CB1) agonist WIN 55, 212-2 mimicked these actions of hypocretin, whereas the application of AM 251, a selective cannabinoid receptor 1 antagonist, depressed the hypocretin-mediated reduction in glutamate transmission. Together, these results are consistent with the idea that postsynaptic activation of hypocretin receptors in serotoninergic dorsal raphe neurons can induce the release of endo-cannabinoids that activate CB1 receptors in presynaptic glutamate terminals, leading to a decrease in transmitter release onto postsynaptic targets (55).

Fig. 7. Hypocretin increases excitatory activity by presynaptic mechanisms. (A) Bath application of hypocretin increased the frequency of miniature excitatory postsynaptic currents (mEPSCs). In the presence of TTX, hypocretin increased the frequency (B), but not the amplitude (C), of mEPSCs recorded in cultured hypothalamic neurons. (Modified from ref. 14.)

Fig. 7. Hypocretin increases excitatory activity by presynaptic mechanisms. (A) Bath application of hypocretin increased the frequency of miniature excitatory postsynaptic currents (mEPSCs). In the presence of TTX, hypocretin increased the frequency (B), but not the amplitude (C), of mEPSCs recorded in cultured hypothalamic neurons. (Modified from ref. 14.)

Fig. 8. Hypocretin enhances GAB A release onto neuroendocrine neurons. (A) Hypocretin increased the frequency of inhibitory synaptic currents in hypothalamic neurons. (B) Experimental paradigm for (C), showing recording pipet, hypocretin-containing pipet, orthodromic electrode to stimulate axons projecting to the recorded cell, and antidromic electrode to stimulate neuroendocrine neurons from their axons in the median eminence ARC, arcuate nucleus. (C) Hypocretin (HCRT) enhanced the GABA-mediated inhibitory-evoked potential (IPSP) in arcuate neurons projecting to the median eminence, suggesting a role for hypocretin in neuroendocrine regulation. (Modified from ref. 14.)

Fig. 8. Hypocretin enhances GAB A release onto neuroendocrine neurons. (A) Hypocretin increased the frequency of inhibitory synaptic currents in hypothalamic neurons. (B) Experimental paradigm for (C), showing recording pipet, hypocretin-containing pipet, orthodromic electrode to stimulate axons projecting to the recorded cell, and antidromic electrode to stimulate neuroendocrine neurons from their axons in the median eminence ARC, arcuate nucleus. (C) Hypocretin (HCRT) enhanced the GABA-mediated inhibitory-evoked potential (IPSP) in arcuate neurons projecting to the median eminence, suggesting a role for hypocretin in neuroendocrine regulation. (Modified from ref. 14.)

3.2. Network Actions of Hypocretin

In addition to its effects on pre- and postsynaptic sites, hypocretin can indirectly modulate the neuronal excitability of a given neuron by acting on the soma of local glutamate or GABA interneurons that make synaptic contacts onto other cells. Voltage clamp recording of serotonin neurons in the dorsal raphe revealed a TTX-sensitive increase in the IPSC frequency with the application of hypocretin, and immunostaining showed hypocretin-containing boutons near the cell body of GABA-expressing cells, consistent with the idea that hypocretin may excite GABA neurons that innervate serotonin neurons (24). Similar spike-dependent indirect actions of hypocretin on GABA interneurons have been observed in the medial septum/diagonal band (23). These hypocretin actions on inhibitory interneurons can serve to prevent overexcitation of neuronal networks due to high levels of hypocretin release.

Hypocretin cells not only send long-distance projections throughout the CNS, but also densely innervate the lateral hypothalamus (14), where they can make contact with the dendrites and cell bodies of local interneurons or with other hypocretin neurons. To evaluate the actions of hypocretin on the cells that produce it, hypocretin-1 or -2 was applied to hypocretin cells in the presence of TTX, but it exerted relatively little effect on their membrane potential. In contrast, in the absence of TTX, hypocretin increased spike frequency, in large part by increasing glutamate release onto hypocretin cells (16). The hypocretin-induced increase in the release of glutamate onto hypocretin neurons may have been due to the direct activation of receptors located in the presynaptic glutamate terminals or attributable to the activation of hypocretin receptors in the somatodendritic region of glutamate neurons innervating hypocretin cells. To address this question, the action of hypocretin on the mEPSCs in the presence of TTX was studied. Under these conditions, hypocretin still increased glutamate release, suggesting that these hypocretin actions were in part caused by activation of its receptors located on presynaptic axons.

The glutamate axons synapsing onto hypocretin neurons could arise from local glutamate interneurons in the LH or from glutamatergic cells elsewhere in the brain. To evaluate this, glutamate microdrop experiments were performed in minislices containing only the lateral hypothalamus (Fig. 9A) (42). Glutamate microdrops, which do not stimulate axons of passage, were applied 1 mm away from the recorded hypocretin cells, and an increase in EPSC frequency was detected in hypocretin neurons (Fig. 9B1). These microdrop actions were completely blocked by TTX (Fig. 9B2), suggesting that they were spike-dependent and not owing to a direct effect of glutamate on the recorded cell (Fig. 9B4). They were also blocked by AP5 and CNQX (Fig. 9B3), confirming that the cells stimulated by the microdrop released glutamate. These results support the idea that glutamate interneurons in the LH innervate hypocretin cells. Some of these LH glutamate interneurons might be activated by hypocretin and could play a role in recruiting the output of a network of dispersed hypocretin neurons. Although most ionotropic glutamate receptor responses to glutamate are excitatory, the group 3 metabotropic glutamate receptor agonist L-AP4 reduced excitatory synaptic activity in hypocretin neurons (Fig. 9C). L-AP4 reduced the frequency of large spike-dependent EPSCs (Fig. 9D), suggesting an action on local glutamate neurons. The mechanism was based on activation of presynaptic metabotropic glutamate receptors on local circuits neurons (42).

Another mechanism by which hypocretin can affect network activity is by modulating timing of discharge of specific cell populations. In the LC, individual neurons may be coupled to others by gap junctions (43), a characteristic that may facilitate the occurrence of coupled oscillations and synchronous firing during directed attention (44). Hypocretin enhances membrane oscillations and synchronous firing in identified noradrenergic LC cells (26). The application of hypocretin-1 to these neurons induced regular oscillations in their membrane potential that were absent in control conditions. In some spontaneously oscillating LC neurons, hypocretin-1 increased the frequency of oscillations by up to 300%. Subthreshold

Fig. 9. (opposite page) Local glutamate interneurons innervate hypocretin neurons. (A) Schematic representation of a lateral hypothalamic (LH) minislice used to study local circuits. (B) Trace 1. Glutamate (Glut) microdrop applied 1 mm away from the recorded cell induced a robust increase in the frequency of excitatory postsynaptic currents (EPSCs) in hypocretin cells. The glutamate microdrop effect was completely abolished by bath application of TTX (trace 2) or by the presence of glutamate receptors antagonists AP5 and CNQX in the bath (trace 3). Direct application of the glutamate microdrop to the recorded hypocretin cell evoked a direct inward current (trace 4). (C,D) The group III metabotropic glutamate receptor agonist LAP-4 reduced synaptic glutamate release onto hypocretin neurons. (Modified from ref. 42.)

Fig. 10. Hypocretin (Hcrt) enhanced the synchronous firing of two locus coeruleus neurons recorded with two electrodes in an LC slice. (Modified from ref. 26.)

membrane potential oscillations might underlie the occurrence of synchronic firing in neurons connected by electrical junctions (45). Using dual cell recording, an increase in the synchrony of spikes was detected with the application of hypocretin to the LC cells (Fig. 10) (26). A similar hypocretin effect has been detected in the spinal cord (19). Here hypocretin increased the amplitude and frequency of membrane potential oscillation and induced synchronous discharge in electrically coupled pairs of spinal neurons previously silent. As hypocretin does not modify the number of coupling junctions in these cells, these results suggest that this peptide directly modulates synchronous firing in neural networks previously silent.

3.3. Many Neurotransmitter Systems Are Excited by Hypocretin

Both hypocretin axons and receptors are widespread within the brain. As hypocretin generally exerts excitatory actions, a number of other transmitter systems would be activated by hypocretin. For instance, the noradrenergic neurons of the LC receive a substantial direct synaptic hypocretin innervation as confirmed with electron microscopy (34), and a number of papers have described different aspects of the excitatory responses in the LC (26,32,46). Thus hypocretin activation of the LC would increase the release probability of norepineph-rine in a wide variety of LC targets. In the lateral hypothalamus, norepinephrine and other catecholamines inhibit the neurons that produce hypocretin (16,56). Hypocretin also activates the serotonin neurons of the dorsal raphe (24,29,33) leading to an increased serotonin release. Similarly, hypocretin activates the serotonin neurons of the dorsal raphe (24,29,33), leading to an increased serotonin release. Similar to the LC neurons, the dorsal raphe has a widespread pattern of axonal innervation. In addition to activation of serotonin neurons, hypocretin also activates local GABAergic neurons in the raphe that terminate on nearby serotonin cells (24).

Hypocretin-1 and -2 excited neurons of the tuberomammillary region identified as hista-minergic by their electrophysiological properties (21,47). Histamine neurons also send axons throughout many regions of the brain (48).

The neurons of the lateral hypothalamus that synthesize melanin-concentrating hormone (MCH) receive a synaptic innervation from hypocretin neurons (49,50). Hypocretin excites MCH neurons by a direct effect on the cell body and by increasing release of glutamate onto MCH neurons (50). Since MCH-containing axons and MCH receptors are found throughout the CNS, activation of the MCH system by hypocretin would increase the probability of MCH release in many different brain regions.

In the spinal cord and brainstem, a number of different neuron types are excited by hypocretin. Hypocretin increases release of the inhibitory amino acid transmitter glycine, and the excitatory transmitter ATP in the dorsal horn of the spinal cord (20). Hypocretin also excites a subset of cells in the nucleus of the solitary tract in the dorsal medulla (40). A subpopulation of neurons in the dorsal medulla synthesize glucagon-like peptide-1, a peptide that exerts multiple excitatory actions on hypocretin-containing cells (57).

Neurons of the medial septum/diagonal band (MSDB) project to a number of sites, with an important projection to the hippocampus. Whereas the MSDB receives a strong hypocre-tin innervation that makes synaptic contact with both GABAergic and cholinergic cells (23,25), the hippocampus itself receives relatively little direct hypocretin innervation (3). Interestingly, hypocretin excites the GABAergic neurons of the MSDB that project to the hippocampus, identified by retrograde transport of tracer injected into the hippocampus back to the recorded neurons (23). These MSDB GABA neurons terminate on inhibitory neurons of the hippocampus. Activation of the MSDB inhibitory cells would lead to an increase in hip-pocampal activity by reducing the inhibitory tone from local hippocampal neurons. Furthermore, hypocretin also excites the cholinergic cells of the MSDB (25), and these cholinergic neurons project directly to the principal neurons of the hippocampus, also leading to excitation. Thus the combination of activating both the GABA and cholinergic cells in the MSDB would lead to a potentially powerful means of activating hippocampal circuits. Hypocretin-1 and -2 excited cholinergic neurons of the basal forebrain but had little effect on the sleep-promoting GABA neurons of the preoptic area (51).

Within the hypothalamus, hypocretin increased the actions of GABAergic neurons that make synaptic contact with neuroendocrine neurons that maintain axons terminating in the median eminence (14). Thus hypocretin would activate an inhibitory circuit that in this case would probably inhibit release of a pituitary tropin into the blood stream of the median eminence. A typical experiment of this sort is shown in Fig. 8B and C, in which a neuroendocrine neuron is identified by antidromic stimulation from the median eminence, and inhibitory projections to the recorded neurons are electrically stimulated. Some neurons in the arcuate nucleus, for instance those that synthesize NPY, receive direct synaptic contact from hypocretin-immunoreactive axons (52) and are excited by hypocretin (53). NPY-containing neurons in the arcuate nucleus and other brain regions send their axons to the lateral hypothalamus where the hypocretin neurons are located; NPY depresses the activity of hypocretin neurons by activation of potassium currents, depression of calcium channels, and reduction in the release of glutamate from presynaptic axons (58).

Hypocretin neurons have a strong projection to the midline thalamic nuclei. Both hypocretin-1 and -2 increased the activity of the centromedial and rhomboid nucleus by a direct action but had no effect on the sensory relay nuclei of the ventral posterolateral or dorsal lateral geniculate (17). The midline thalamic nuclei maintain a nonspecific cortical projection, which may enhance cortical activation.

In summary, neurons in many regions of the brain show excitatory responses to hypocretin. Multiple pre- and postsynaptic mechanisms underlie hypocretin's actions. These mechanisms are owing to complex signal transduction pathways that couple hypocretin receptor activation, primarily through Gq protein, with changes in Na+, K+, and Ca2+ ion channel conductance.

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